PROJECT SIGNIFICANCE: Spindle orientation is critical during asymmetric cell division and is important to a wide range of developmental processes in systems ranging from yeast to man. The minus-end directed microtubule motor dynein plays a pivotal role in spindle orientation during asymmetric cell division in numerous organisms. Central to the role of dynein in spindle orientation is how dynein is anchored at the cortical membrane. This is not well understood in any system. OBJECTIVE/HYPOTHESIS: The goal of this proposal is to investigate how dynein interacts with cortical receptor proteins to mediate spindle orientation. The budding yeast provides an excellent model system for defining dynein-cortex interactions during spindle orientation because dynein only function in yeast is to position the mitotic spindle. Unlike many cell types where large numbers of astral microtubules interact with the cortex at many cortical sites for spindle orientation, in yeast, dynein exerts cortical pulling forces on a single cytoplasmic microtubule. Yeast is also the only system in which a candidate cortical receptor protein for dynein has been identified. This receptor, Num1, localizes to discrete patches at the cortex. My work supports a model in which dynein uses the microtubule plus end to reach the edge of the cell and off-load onto cortical patches composed of a Num1 receptor complex. Off-loaded dynein becomes anchored to generate pulling forces for spindle positioning. SPECIFIC AIMS: (1) To test the off-loading model by directly imaging the process using time-lapse fluorescence microscopy. (2) To use biochemical and candidate gene strategies to identify proteins that bridge dynein at the plus end to Num1 patches, as well as proteins that are responsible for cortical patch assembly. (3) To test the function of Num1 patch formation and identify the determinants for patch assembly using mutant and deletion constructs of Num1;to use yeast mutants to probe the role of phospholipids in Num1 patch localization. RELEVANCE TO PUBLIC HEALTH: Understanding the molecular mechanism of spindle positioning is highly significant because correct spindle orientation is critical for maintaining genomic stability and plays a role in stem cell differentiation and development in humans.